This Bioengineering Research Partnership will develop biomolecular analysis and design tools for engineering next-generation therapeutic antibodies for cancer therapies. Generating antibodies to bind targets whose expression is increased in tumor cells has been a promising therapeutic approach for many years. However, antibody binding can produce a variety of different cellular responses and, ultimately, control over tumor cells will require careful study of the relationship between antibody binding and cellular response so that appropriate binding properties can be engineered to produce desired responses. In the research proposed here, antibodies against epidermal growth factor receptor (EGFR) will be isolated and engineered by directed evolution, guided by computational docking predictions and quantitative analysis of cellular responses to altered EGFR signaling, dimerization, and trafficking. EGFR is overexpressed in a broad spectrum of epithelial cancers, and blocking antibodies are currently progressing favorably through clinical immune response and limitations of screening assays rather than rational design for these initial antibody drugs, and consequently the molecular mechanism of efficacy in vivo is not fully understood. Targeting specific EGFR surfaces with antibodies designed to maximize receptor antagonism and downregulation should result in greater therapeutic efficacy. The proposed research will develop approaches applicable to antibody design against a broad range of targets, particularly cell surface receptors. The BRP team integrates expertise in protein engineering, cellular bioengineering, computational protein biophysics candidates; generic tools for engineering protein/protein recognition; and will develop analytical approaches to biological response modification via combined quantitative cell biology and protein engineering.